Regulatory T cell (Treg) plays a critical role in maintaining immune system homeostasis and preventing autoimmunity and immunopathology. Compromised Treg function is linked to multiple autoimmune diseases. The development and maintenance of Treg cell lineage is dependent on transcription factor Foxp3, as loss of function mutations lead to severe lymphoproliferative disease in mice and humans. Thus, understanding the mechanisms that govern Foxp3 induction and stability may lead to novel therapies for autoimmune disease. Using a comparative genomic approach, Dr. Zheng and colleagues identified three evolutionarily conserved intronic cis-elements at the Foxp3 locus (conserved non-coding sequences (CNS) 1, 2 and 3). To elucidate the role of these CNS regions in Treg lineage development in vivo, they proceeded to knockout individual Foxp3 CNS regions in mice. Each of the three CNS knockout mice showed different defects in their Treg population, indicating the unique roles these CNS regions play in regulating Foxp3 expression. CNS2 is unique as it is located in the only CpG island at the Foxp3 locus. The methylation status of CNS2 is one of the most prominent epigenetic markers distinguishing Tregs from conventional T cells. Induction of Foxp3 is not affected by CNS2 deletion, but stabilization of Foxp3 expression is compromised. The mechanism underlying Treg lineage stability is not only a central question in the Treg biology field, but also has practical implications in Treg cell based therapy for autoimmune disease and organ transplant patients. The CNS2 KO mice provide a unique opportunity to explore the molecular and cellular mechanisms of Treg lineage stability in vitro and in vivo. Dr. Zheng's group will study the role of CNS2 in long-term maintenance of Treg stability in vivo, and determine if ex-Tregs are a major driver of autoimmunity (Aim 1). Additionally, they will address how Treg lineage stability is maintained under inflammation, a critical checkpoint for development of autoimmunity (Aim 2). They will dissect the molecular pathways that are involved in safeguarding Tregs under inflammatory conditions, which can potentially lead to drug targets for stabilizing Tregs for therapeutic purpose. Furthermore, Dr. Zheng's group will employ state-of-the-art mouse genetic tools to study the lineage stability of gut commensal induced iTregs (Aim 3). Since Treg lineage stability is directly related to the safety of Treg cell- based therapy for a variety of immune diseases, Dr. Zheng's study on CNS2 will provide insight on an important regulatory circuitry safeguarding Treg identity.